The invention relates to methods of manipulating gene expression in plants.
The ability to manipulate gene expression provides a means of producing new characteristics in transformed plants. There are many situations in which high or increased levels of gene expression may be desired. For example, it is desirable to increase production of a protein that itself maximizes the disease resistance, yield, flavor, or any other commercially important attribute of a plant. Similarly, the regulation of endogenous gene expression by the exogenous expression of antisense, ribozyme RNA, or transgene silencing may result in more valuable plants or plant products. The enhancement of expression, through the use of the invention disclosed herein, would facilitate these possibilities.
We have discovered duplicated enhancer domains for use in the enhancement of gene expression in transgenic plants. The duplicated enhancer domains have a plurality of the repetitive units of one or more enhancers derived from cassava vein mosaic virus (CsVMV). The duplicated enhancer domains are preferably accompanied by a promoter that includes an RNA polymerase binding site and an mRNA initiation site. Expression constructs, including a duplicated enhancer domain and a promoter, provide for enhanced expression of a desired trait compared to that achieved with the promoter in the absence of the duplicated enhancer domain.
Accordingly, in a first aspect, the invention features an enhancer cassette that includes a duplicated enhancer domain derived from a cassava vein mosaic virus.
Preferably, the enhancer cassette includes a component having the formula (X-Y)n, wherein X corresponds to the enhancer domain derived from a cassava vein mosaic virus, Y is an intervening spacer domain having a sequence that is placed between enhancer domains and is typically between about zero and about five hundred nucleotides inclusive (preferably between zero and about one hundred nucleotides and, more preferably, between zero and thirty nucleotides), and n is an integer between 2 and 8 inclusive. In preferred embodiments, the enhancer domain (X) has a sequence that includes nucleotides 1 to about 261 of SEQ ID NO: 1, nucleotides 1 to about 332 of SEQ ID NO: 1, or nucleotides of about 262 to about 332 of SEQ ID NO: 1. Preferably, in the formula of the first aspect, (X-Y)n, n is 2. The spacer domains (Y) can be identical or different. For example, an enhancer cassette having the formula (X-Y)3 can have three different spacer sequences.
In a related aspect, the invention features an expression construct including the enhancer cassette of the first aspect and a second component that includes a promoter having an RNA polymerase binding site and an mRNA initiation site. A preferred promoter is a cassava vein mosaic virus promoter, such as one included in the nucleotides from about 333 to about 444 of SEQ ID NO: 1. The promoter can also be a heterologous promoter (for example, a Ti-plasmid promoter such as the T-DNA gene 5 or 7 promoter). In preferred embodiments, the expression construct includes a sequence corresponding to SEQ ID NO: 2. (FIG. 4A), SEQ ID NO: 3 (FIG. 4B), SEQ ID NO: 4 (FIG. 4C), SEQ ID, NO: 5 (FIG. 4D), or SEQ ID NO: 6 (FIG. 4E).
The expression construct may further include, as a third component, a nucleic acid molecule of interest, wherein the first, second, and third components are operably linked so that the nucleic acid molecule is transcribed. The third component of the construct can encode a protein providing disease or insect resistance, an antisense RNA, a selectable marker (e.g., GUS, GFP, and the like), a non-translatable RNA molecule, or any protein or RNA that improves or results in a desired attribute.
This three-component expression construct of the present invention, when placed in a transcription medium capable of supporting transcription, typically results in increased transcription of the nucleic acid molecule relative to transcription of the nucleic acid molecule operably linked to an expression construct that has only one CsVMV enhancer domain.
In related aspects, the invention also features vectors and cells that include the enhancer cassette of the first aspect. Preferably, the cell is a eukaryotic cell, and, more preferably, a plant cell (e.g., from a monocotylenous plant or a dicotylenous plant).
In another related aspect, the invention also features a transgenic plant that includes the enhancer cassette of the first aspect.
In yet another related aspect, the invention features a method for expressing a nucleic acid molecule. The method includes transforming a cell (for example, a plant cell) with an expression construct that includes (a) a first component having the formula (X-Y)n, wherein X corresponds to the enhancer domain derived from a cassava vein mosaic virus, Y is an intervening spacer domain having a sequence that is placed between enhancer domains and is typically between about zero and about five hundred nucleotides inclusive (preferably between zero and about one hundred nucleotides and, more preferably, between zero and thirty nucleotides), and n is an integer between 2 and 8 inclusive; (b) a second component that includes a promoter (e.g., an RNA polymerase binding, site and an mRNA initiation site); and (c) a third component that includes the nucleic acid molecule to be expressed, wherein the first, second, and third components are operably linked so that the nucleic acid molecule is transcribed. In preferred embodiments, the enhancer domain (X) consists of a sequence that includes nucleotides from about 1 to about 261 of SEQ ID NO: 1, nucleotides from about 1 to about 332 of SEQ ID NO: 1, and nucleotides from about 262 to about 332 of SEQ ID NO: 1. Preferably, in the formula of the third aspect, (X-Y)n, n is 2.
The promoter can be any promoter that is functional in the transformed cell, but preferably is a cassava vein mosaic virus promoter (e.g. one included in nucleotides from about 333 to about 444 of SEQ ID NO: 1).
The third component of the expression construct can encode a protein providing disease or insect resistance, an antisense RNA, a selectable marker (e.g., GUS, GFP, and the like), or any protein or RNA including but not limited to a nontranslatable RNA or any RNA molecule capable of inducing transgene silencing.
As used herein, by xe2x80x9cnucleic acidxe2x80x9d is meant either DNA or RNA. A xe2x80x9cnucleic acid moleculexe2x80x9d may be a single-stranded or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Unless otherwise specified, the left hand direction of the sequence of a single-stranded nucleic acid molecule is the 5xe2x80x2 end, and the left hand direction of double-stranded nucleic molecule is referred to as the 5xe2x80x2 direction.
By xe2x80x9cpromoterxe2x80x9d is meant a region of nucleic acid, upstream from a translational start codon, which is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A xe2x80x9cplant promoterxe2x80x9d is a promoter capable of initiating transcription in a plant cell, and may or may not be derived from a plant cell. A xe2x80x9cCsVMV promoterxe2x80x9d is one derived from the promoter region of a CsVMV genome and that, when operably linked to a heterologous. nucleic acid molecule, is capable of initiating transcription of that molecule when present in a transcription medium capable of supporting transcription, such as in a plant cell, a plant, or in vitro.
Exemplary transcription media include, for example, a plant cell, plant protoplasts, or other plant tissue culture configurations, non-differentiated plant cells, differentiated plant cells (such as cultured plantlets), transgeinic plants, and mature plants. Also included are in vitro expression systems such as reconstituted expression medium composed of components required to support transcription, as are known in the art.
By xe2x80x9cenhancer domainxe2x80x9d is meant a nucleic acid sequence that, when positioned proximate to a promoter and present in a transcription medium capable of supporting transcription, confers increased expression relative to the expression resulting from the promoter in,the absence of the enhancer domain. By xe2x80x9cenhancer cassettexe2x80x9d is meant a nucleic acid sequence that includes an enhancer domain and, optionally, additional sequence that does not enhance expression (e.g; intervening spacer domain).
By xe2x80x9cduplicated enhancer domainxe2x80x9d is meant two or more copies of an enhancer domain. Preferably, the number of copies is between about two and about four. The enhancer domains can be in the same or opposite orientation, and can be contiguous or noncontiguous. In the case of expression constructs having two duplicated enhancer domains (e.g., domain A and domain B), the orientation and the 5xe2x80x2 to 3xe2x80x2 order (e.g., 5xe2x80x2-AABB-3xe2x80x2 vs. 5xe2x80x2-ABAB-3xe2x80x2) are not limitations to the invention. The enhancer domains may also be separated by intervening spacer domains as described herein.
By xe2x80x9cOperably linkedxe2x80x9d is meant that a nucleic acid molecule to be transcribed and an expression construct (i.e., a promoter and an enhancer domain) are connected in such a way as to permit transcription of the nucleic acid molecule in a suitable transcription medium.
By xe2x80x9cderived fromxe2x80x9d is meant that the nucleic acid molecule was either made or designed from a second nucleic acid molecule, the derivative retaining the functional features thereof.
By xe2x80x9cexpression constructxe2x80x9d is meant a nucleic acid molecule that is capable of directing transcription. An expression construct of the present invention includes, at the least, a duplicated CsVMV enhancer domain and a promoter. Additional domains, such as a transcription termination signal, may also be included, as described herein.
By xe2x80x9cvectorxe2x80x9d or xe2x80x9cexpression vectorxe2x80x9d is meant an expression system, a nucleic acid-based shuttle vehicle, a nucleic acid molecule adapted for nucleic acid delivery, or an autonomous self-replicating circular DNA (e.g., a plasmid). When a vector is maintained in a host cell, the vector can either be stably replicated by the cells during mitosis as an autonomous structure, incorporated within the genome of the host cell, or maintained in the host cell""s nucleus or cytoplasm.
By xe2x80x9cplasmidxe2x80x9d is meant an autonomous DNA molecule capable of replication in a cell, and includes both expression and nonexpression types.
By xe2x80x9cheterologousxe2x80x9d is meant that the nucleic acid molecule originates from a foreign source or, if from the same source, is modified from its original form or sequence. Thus, a xe2x80x9cheterologous promoterxe2x80x9d is a promoter not normally associated with the enhancer domain that is duplicated. Similarly, a heterologous nucleic acid molecule that is modified from its original form or is from a source different from the source from which the promoter to which it is operably linked was derived.
The term xe2x80x9cplantxe2x80x9d includes any cell having a plastid, and can include whole plants, plant organs (e.g., stems, leaves, roots, etc.), seeds, and cells. The class of plants that can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocots and dicots.
By xe2x80x9ctransgenexe2x80x9d is meant any piece of a nucleic acid molecule (for example, DNA) which is inserted by artifice into a cell, and becomes part of the organism (integrated into the genome or maintained extrachromosomally) which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
By xe2x80x9ctransgenic plantxe2x80x9d is meant a plant containing a transgene. For example, a plant cell transformed with a vector containing the expression construct of the present invention operably linked to a heterologous nucleic acid molecule can be used to produce a transgenic plant having altered phenotypic characteristics.